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Purification of bacteriophage lambda repressor.

ABSTRACT: Bacteriophage lambda repressor controls the lysogeny/lytic growth switch after infection of E. coli by lambda phage. In order to study in detail the looping of DNA mediated by the protein, tag-free repressor and a loss-of-cooperativity mutant were expressed in E.coli and purified by (1) ammonium sulfate fractionation, (2) anion-exchange chromatography and (3) heparin affinity chromatography. This method employs more recently developed and readily available chromatography resins to produce highly pure protein in good yield. In tethered particle motion looping assays and atomic force microscopy "footprinting" assays, both the wild-type protein and a C-terminal His-tagged variant, purified using immobilized metal affinity chromatography, bound specifically to high affinity sites to mediate loop formation. In contrast the G147D loss-of-cooperativity mutant bound specifically but did not secure loops.

SUBMITTER: Gao N

PROVIDER: S-EPMC3761395 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Purification of bacteriophage lambda repressor.

Gao Ning N Shearwin Keith K Mack John J Finzi Laura L Dunlap David D

Protein expression and purification 20130703 1

Bacteriophage lambda repressor controls the lysogeny/lytic growth switch after infection of E. coli by lambda phage. In order to study in detail the looping of DNA mediated by the protein, tag-free repressor and a loss-of-cooperativity mutant were expressed in E.coli and purified by (1) ammonium sulfate fractionation, (2) anion-exchange chromatography and (3) heparin affinity chromatography. This method employs more recently developed and readily available chromatography resins to produce highly ...[more]

PMID: 23831434

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Project description:The first-order rate constants for the RecA-independent, spontaneous, pH-dependent autocleavage of the lambda cI repressor was measured in the present study at pH 10.6 at 27, 37 and 42 degrees C respectively. Autocleavage of the repressor occurs also at pH 9 and 8, although at progressively slower rates. We demonstrate that the spontaneous autocleavage occurs also in the operator-bound state, at a rate either higher than or equal to the rate in solution, depending on the pH value. Owing to the near equality of the rate constant in both operator-free and operator-bound repressors, it can be inferred that the cleavage site has a similar structure and dynamics with respect to the catalytic site in both forms at neutral pH. Covalent modification using PMSF, brought about by a large molar excess of the reagent, inhibits autocleavage of the lambda repressor. The difficulty in obtaining this covalent modification is rationalized using our recent lambda repressor models. Bimolecular type II trans -cleavage was observed previously for mutant LexA repressors lacking a crucial catalytic serine or lysine residue [Kim and Little (1993) Cell (Cambridge, Mass.) 73, 1165-1173], but it could still be cleaved by an 85-202 'enzyme' fragment possessing an improved or hypercleavable character lacking its own cleavage site. Such a type II trans -cleavage was not observed with the covalently modified intact lambda repressor used as substrate and the purified wild-type lambda repressor 112-236 fragment used as the 'enzyme'. All these results show that for the wild-type lambda repressor, the catalytic site is close to the cleavage site in both operator-free and -bound states. In the lytic pathway, the repressor is mainly cleaved via RecA-mediated cleavage, which occurs much faster than the spontaneous autocleavage; the possible biological significance of this slow, spontaneous, but constant, autocleavage is related to the lysogenic state, when RecA-mediated cleavage is absent.

Dataset Information

Purification of bacteriophage lambda repressor.

Publications

Purification of bacteriophage lambda repressor.

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